The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants plus those of S. pombe to elucidate the physiological functions of the polyamines, and, in particular, to ascertain the physiological effects of polyamine deprivation. In our last report we reported our studies on the effects of polyamine-deprivation on cell-cycle progression in S. pombe; we showed that in the absence of polyamines the cell cycle stops in the G1-S phase, and that these cells showed profound morphological changes. During the current year we have completed our studies showing that polyamines are important in protecting E. coli cells from the toxic effects of oxygen and hydrogen peroxide. In these studies we showed that, whereas wild type cells can be grown in an 100% oxygen atmosphere, polyamine-deficient cells are killed under these conditions. Addition of polyamines to the medium prevents this toxicity. The involvement of superoxide in this toxicity was shown by the ability of the Mn-SOD plasmid to protect the polyamine-deficient cells from the oxygen toxicity. Partial protection was also achieved by the addition of certain amino acid mixtures or by addition of sucrose or sorbitol. In our most recent work we have studied the relative importance of spermidine and spermine for the growth of S. cerevisiae. In these studies we have shown that spermidine is specifically required for the growth of S. cerevisiae, and that spermine will not substitute for this requirement unless it is first converted to spermidine by the FMS1 encoded amine oxidase. In these in vivo studies we have shown that in yeast, as in other cells, spermidine, and not spermine, is required for the hypusine modification of the protein initiation factor eIF5a, and that presumably this is one reason for the specific requirement of spermidine for growth. In addition to the in vivo studies we have purified the FMS1 encoded amine oxidase from an overexpressed yeast strain, and studied the amine oxidase activity of the purified protein in vitro. Current work is directed at extending the use of the spe3 ( delta) fms1 (delta) mutants to elucidate the relative importance of spermidine and spermine in a variety of physiological functions.
